Catalytic activation of multimeric RNase E and RNase G by 5′-monophosphorylated RNA
Catalytic activation of multimeric RNase E and RNase G by 5′-monophosphorylated RNA
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DOI：
10.1073/pnas.0401382101
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发表时间：
2004-06-22
影响因子：
11.1
通讯作者：
Belasco, JG
中科院分区：
文献类型：
--
作者：
Jiang, XQ;Belasco, JG
RNase E 是一种核酸内切酶，在大肠杆菌的 RNA 加工和降解中发挥着核心作用。与它的大肠杆菌同源物 RNase G 一样，RNase E 显示出明显偏向于切割 5' 端带有单磷酸盐的 RNA，而不是带有三磷酸盐或羟基的 RNA。为了研究 5' 末端磷酸化影响远距离裂解事件的机制，我们开发了荧光 RNA 底物，可以比以前更准确、更轻松地定量 RNase E 和 RNase G 的活性。对 RNase E 和 RNase G 裂解这些底物的动力学分析表明，5' 单磷酸化不是通过改善底物结合来加速反应，而是通过增强这些核糖核酸酶的催化效力。此外，5'单磷酸的存在可以增加RNA内切割位点选择的特异性。尽管单体形式的 RNase E 和 RNase G 可以切割 RNA，但这些酶根据 5' 磷酸化状态区分 RNA 底物的能力需要形成蛋白质多聚体。可以解释这些特性的分子机制包括一个酶亚基的 5' 端结合诱导蛋白质结构变化，从而加速另一个亚基对 RNA 的切割。
RNase E is an endonuclease that plays a central role in RNA processing and degradation in Escherichia coli. Like its E. coli homolog RNase G, RNase E shows a marked preference for cleaving RNAs that bear a monophosphate, rather than a triphosphate or hydroxyl, at the 5' end. To investigate the mechanism by which 5'-terminal phosphorylation can influence distant cleavage events, we have developed fluorogenic RNA substrates that allow the activity of RNase E and RNase G to be quantified much more accurately and easily than before. Kinetic analysis of the cleavage of these substrates by RNase E and RNase G has revealed that 5' monophosphorylation accelerates the reaction not by improving substrate binding, but rather by enhancing the catalytic potency of these ribonucleases. Furthermore, the presence of a 5' monophosphate can increase the specificity of cleavage site selection within an RNA. Although monomeric forms of RNase E and RNase G can cut RNA, the ability of these enzymes to discriminate between RNA substrates on the basis of their 5' phosphorylation state requires the formation of protein multimers. Among the molecular mechanisms that could account for these properties are those in which 5'-end binding by one enzyme subunit induces a protein structural change that accelerates RNA cleavage by another subunit.